`Eng.Eng.
`
`LifeLife
`Capillary IP-RP-HPLC
`
`Sci.Sci.
`Polystyrene/Divinylbenzene Based Monolithic and Encapsulated
`Capillary Columns for the Analysis of Nucleic Acids by
`High-Performance Liquid Chromatography-Electrospray
`Ionization Mass Spectrometry
`
`By C. W. Huck*, R. Bakry, and G. K. Bonn
`
`Monolithic capillary columns are prepared by copolymerization of styrene and divinylbenzene, encapsulated capillary col-
`umns by immobilizing silica particles with different pore sizes inside a 200 lm i.d. fused silica capillary by encapsulation of
`the derivatized silica sorbent in a poly(styrene/divinylbenzene) (PS/DVB) matrix. Both allow the rapid and highly efficient
`separation of single- and double-stranded DNA by ion-pair reversed-phase high-performance liquid chromatography
`(IP-RP-HPLC). The high resolving power of monolithic and encapsulated capillary columns can be utilized for mutation
`screening in polymerase chain reaction (PCR) amplified polymorphic loci by denaturing HPLC (DHPLC). Recognition of
`mutations is based on the separation of homo- and heteroduplex species by IP-RP-HPLC under denaturing conditions,
`resulting in characteristic peak patterns both for homozygous and heterozygous samples. Separations can be readily hyphe-
`nated to electrospray ionization-mass spectrometry.
`
`matches by IP-RP-HPLC at elevated column temperatures
`(48±67 C). The low flow rates are highly suited for on-line
`hyphenation to mass spectrometry via an electrospray ion-
`ization interface (ESI), which has become an important tool
`in the structural analysis of nucleic acids [18]. Therefore, IP-
`RP-HPLC offers the possibility to use volatile mobile phases
`of low ionic strength in order to ensure high compatibility
`with the ESI-process. For the separation of biological macro-
`molecules, such as nucleic acids and PCR products station-
`ary phases with favourable mass transfer properties are re-
`quired.
`Since the introduction of micro particular stationary
`phases in 1967 [19] a general problem is the slow mass trans-
`fer of solutes into and out of the stagnant mobile phase pres-
`ent in the micro- and mesopores of the stationary phase,
`which results in considerable band broadening particularly
`with high molecular analytes such as DNA [20, 21]. There
`are five different ways to enhance the mass transfer of bio-
`molecules in stationary phases:
`1. increasing the ratio of pore diameter to particle diameter
`[22];
`2. using non-porous stationary phases to eliminate support
`pores [23];
`3. decreasing the pore depth [24];
`4. introducing flow-through pores into the stationary phase
`particle [25]; and finally
`5. using porous monoliths or silica encapsulated monoliths
`as the chromatographic bed [26±28].
`Monolithic and encapsulated column configuration lacking
`diffusive micro- and mesopores may be adequately described
`as a micropellicular phase [29] and have been shown to en-
`able the separation of analytes over a very broad size range
`with efficiencies significantly better compared to that of col-
`umns packed with micropellicular granular stationary phases.
`The fact is that all of the mobile phase is forced to flow
`
` 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`431
`
`1 Introduction
`
`Due to the high importance of nucleic acids in biosciences
`there is a permanent demand for the development of highly
`efficient and fast techniques for their isolation, separation,
`quantitation, and structural analysis. Separation of nucleic
`acid mixtures can be utilized in six modes using high-perfor-
`mance liquid chromatography (HPLC), including size-exclu-
`sion chromatography [1], anion-exchange HPLC [2,3],
`mixed-mode HPLC [4], reversed-phase HPLC (RP-HPLC),
`ion-pair reversed-phase HPLC (IP-RP-HPLC) [5], and af-
`finity chromatography [6]. Most commonly anion-exchange
`HPLC and IP-RP-HPLC are applied because of their high
`resolution capability and flexibility to separate both single-
`and double-stranded nucleic acids in a size range from a few
`nucleotides to several thousand base pairs. Methods used for
`the detection of unknown mutations include single strand
`conformation polymorphism analysis [7], denaturing gradi-
`ent gel electrophoresis [8], temperature gradient gel electro-
`phoresis [9], mismatch cleavage methods [10, 11], direct
`sequencing [12], oligonucleotide arrays [13], as well as gel
`electrophoresis [14] and high-performance liquid chroma-
`tography-based [15] heteroduplex detection.
`Denaturing high-performance liquid chromatography
`(DHPLC) shows high potential in the detection of single-
`base substitutions and small deletions or insertions in DNA
`ranging from 100 to 1500 base pairs [16, 17]. DHPLC is
`based on the separation of homo- from heteroduplex species
`generated by polymerase chain reaction (PCR) ampli-
`fication of polymorphic loci containing one or more mis-
`
`± [
`
`*] C. W. Huck (christian.w.huck@uibk.ac.at), R. Bakry, G. K. Bonn, Institute
`of Analytical Chemistry and Radiochemistry, Leopold-Franzens-Uni-
`versity, Innrain 52a, A-6020 Innsbruck, Austria.
`
`Eng. Life Sci. 2005, 5, No. 5
`
`DOI: 10.1002/elsc.200520110
`Please note: corrected DOI, in print
`wrong DOI (10.1002/elsc.20050063)
`
`1
`
`MTX1009
`
`

`

`Eng.
`Life
`Sci. Full Paper
`
`through the pores of the separation medium because of the
`absence of an intra-particular volume [30], which means an
`enhancement of mass transport by such convection [31, 32].
`Monolithic columns can be synthesized by packing the col-
`umn with granular stationary phases followed by sintering or
`cross-linking of the micro particles [33], or by polymerization
`or polycondensation of suitable monomers and porogens in a
`stainless steel or fused silica tube [26, 27]. Encapsulated cap-
`illary columns are prepared by immobilizing derivatized sili-
`ca particles with different pore diameters within a fused silica
`capillary by encapsulation of the silica sorbent in a poly(sty-
`rene/divinylbenzene) (PS/DVB) matrix. The usefulness of
`this method arises from the specific advantage of the ease in
`which capillary columns can be prepared with selected func-
`tionalized chromatographic materials and the possibility to
`analyze both low- and even high-molecular biomolecules
`[34]. In the preparation of both phases, a porous structure is
`achieved during the polymerization procedure due to the
`presence of a monomer or monomer mixture containing ap-
`propriate amounts of both a cross-linking monomer and a
`porogenic solvent or a mixture of porogenic solvents [35].
`Due to the immobilization of the PS/DVB monolith at the
`capillary wall the necessity to prepare tiny retaining frits can
`be cancelled. Miniaturized chromatographic separation sys-
`tems applying capillary columns of 10±500 lm i.d. are fre-
`quently the method of choice for DNA analysis, especially
`when the amount of sample is limited. Monolithic and en-
`capsulated capillary columns have been proven to be effi-
`cient for the separation of peptides [36, 37] and proteins
`[38]. Monolithic separation media derivatized with anion-ex-
`change functional groups can successfully be applied to the
`separation of mononucleotides [39], oligonucleotides [40, 41],
`and plasmid DNA [42]. In this contribution highly efficient
`separations of single-stranded oligonucleotides and double-
`stranded DNA fragments over a range of five nucleotides to
`622 base pairs as well as the detection of single nucleotide
`
`polymorphisms (SNPs) in genomic DNA using monolithic
`and encapsulated poly(styrene/divinylbenzene) based capil-
`lary columns are considered. The miniaturized separation
`system is on-line hyphenated to ESI-MS, which allows accu-
`rately characterizing the masses.
`
`2 Properties of Micropellicular, Monolithic and
`Encapsulated Separation Media
`
`For the separation of large biomolecules having low diffusiv-
`ities stationary phase structures that allow favourable mass
`transfer are required. One approach is the use of micropelli-
`cular configurations consisting of a core of fluid-impervious
`support material covered by a thin retentive layer of station-
`ary phase [23, 43]. A second approach to alleviate the mass-
`transport problem is the usage of a multimodal pore size
`distribution, where macro-pores ensure the flow through the
`support material while meso- and micro-pores form the sur-
`face for chromatographic interaction. This concept can be
`applied by the use of perfusion particles [44] and monolithic
`stationary phases [26, 45].
`The advantage of enhanced mass-transfer properties can
`be realized by the combination of both the micropellicular,
`monolithic, and encapsulated configuration. For the prepara-
`tion of poly(styrene/divinylbenzene)-based monoliths as well
`as encapsulated capillary columns the use of 1-decanol as
`macro- and meso-porogen favours the formation of macro-
`pores and suppress the formation of micro-pores [45]. This
`results in stationary phases owing micropellicular structures.
`Fig. 1 illustrates the structural differences between a col-
`umn packed with particles (see Fig. 1a), one packed with a
`monolith (see Fig. 1b) and an encapsulated capillary column
`(see Fig. 1c). Conventional columns are filled with tightly
`packed particles (see Fig. 1a), which allow only a very weak
`intra-particular mass-transfer. The monolithic column con-
`
`+
`
`Styrene
`
`AIBN
`
`70ºC
`24 h
`
`Divinylbenzene
`
`Dioxan/Decanol
`
`Polystyrene/Divinylbenzene
`
`a
`
`b
`
`c
`
`5 µm
`
`5 µm
`
`5 µm
`
`5 µm
`
`5 µm
`
`5 µm
`
`Figure 1. Structural characteristics of (a) packed, (b) monolithic, and (c) encapsulated chromatographic beds.
`
`432
`
` 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`http://www.els-journal.de
`
`Eng. Life Sci. 2005, 5, No. 5
`
`2
`
`

`

`Capillary IP-RP-HPLC
`
`
`Eng.Eng.
`
`LifeLife
`
`Sci.Sci.
`
`of 200 lm were used. Comparison of the peak width at half
`height shows that using both the monolithic and encapsu-
`lated 200 lm i.d. connected to a 3 nL capillary detection cell
`allow the determination at halved peak width (values be-
`tween 2.2 and 2.8 s) rather than using the micropellicular
`packed 2 mm i.d. column (values between 4.2 and 4.9 s) re-
`sults apart from the lower injected sample volume in higher
`resolution.
`
`3 Analysis of Single-stranded Oligonucleotides
`
`For the separation of oligonucleotides by ion-pair reversed-
`phase HPLC amphiphilic ions such as triethylammonium
`ions are added to the hydro organic phase, which is mostly
`comprised of water and acetonitrile. This forms a positive
`surface potential for the retention of negatively charged ana-
`lytes such as nucleic acids, which are then eluted by a gradi-
`ent of increasing acetonitrile concentration [49].
`The resolving power of a monolithic capillary column for
`the separation of single-stranded oligonucleotides by IP-RP-
`HPLC is demonstrated in Fig. 3 upon the separation of a
`ladder of mixed-sequence single-stranded oligonucleotides
`ranging in size from eight to 32 nucleotide units and differ-
`ing in length by two nucleotide units [50]. The resolution val-
`ues range from 7.09 between the 8-mer and the 10-mer to
`2.69 between the 30-mer and the 32-mer, and the peak con-
`centration is proportional to the inverse square of the col-
`umn diameter. Comparing capillary to conventional HPLC
`the signals are 500 times higher than applying a 4.6 mm i.d.
`This results in a tenth of injection volumes with the detec-
`tion limit being as low as 3 fmol for an 18-mer oligonucleo-
`tides using UV-absorbance detection at 254 nm. This is al-
`most as sensitive as fluorescence detection of fluorescent
`dye-labelled nucleic acids using a 4.6 mm i.d. column, with
`
`sists of one single porous solid with relatively large channels
`for convective flow (see Fig. 1b) and additionally it consists
`of no micro-pores [46]. This phase configuration allows im-
`proving the transport of the analytes within the packed col-
`umn, which can also be calculated by a mathematical model
`[47]. Silica encapsulated stationary phases (see Fig. 1c) are
`characterized similar to monoliths by a predominant number
`of macro- and a small content of meso-pores. Comparing the
`electron micrographs in Fig. 1 it is obvious that monolithic
`and encapsulated capillary columns have a higher number of
`channels penetrating the chromatographic bed than columns
`packed with particles.
`The chromatographic performance of the three stationary
`phases is compared in Fig. 2 upon injection of a 12-mer oli-
`godeoxythymidylic acid pd(T)12±18. In Fig. 2a a semi-micro-
`column with an i.d. of 2 mm packed with micropellicular par-
`ticles, in Figure 2b a monolithic capillary column and in
`Fig. 2c an encapsulated capillary column both having an i.d.
`
`
`
`4.2 s4.2 s
`
`pd(T)18
`pd(T)18
`pd(T)17
`pd(T)17
`
`pd(T)16
`pd(T)16
`
`pd(T)15
`pd(T)15
`
`pd(T)14
`pd(T)14
`
`pd(T)13
`pd(T)13
`
`pd(T)12
`pd(T)12
`
`aa
`
`
`
`4.9 s4.9 s
`
`5
`
`7
`
`9
`
`11
`
`13
`
`
`
`2.8 s2.8 s
`
`
`
`2.5 s2.5 s
`
`4
`
`5
`
`6
`
`7
`
`
`
`2.4 s2.4 s
`
`
`
`2.2 s2.2 s
`
`bb
`
`3
`
`cc
`
`2
`
`3
`
`4
`time [min]
`
`5
`
`6
`
`Figure 2. Comparison of the chromatographic performance of (a) a column
`packed with micropellicular particles, (b) a monolithic, and (c) an encapsulated
`column for the separation of oligonucleotides. Column (a) 50 ” 2 mm i.d.,
`packed with 2.3 lm PS/DVB-C18 particles, (b) 50 ” 0.2 mm i.d., PS/DVB mono-
`lith, (c) 50 ” 0.2 mm i.d., PS/DVB encapsulated Si-C18 (5 lm, 80 Š); mobile
`phase: (A) 100 mM TEAA, pH 7.00, (B) 100 mM TEAA, pH 7.00, 20 % aceto-
`nitrile; linear gradient: (a) 20±50 % B in 15 min, (b) and (c) 25±75 % B in
`10 min; flow rate: (a) 200 lL/min, (b) 3.0 lL/min, (c) 5.0 lL/min; detection: UV
`254 nm; sample: (a) 25 ng, (b) and (c) 2.5 ng pd(T)12±18.
`
`Figure 3. Chromatogram of mixed sequence oligonucleotide sizing markers
`eight to 32 bases in length separated using a monolithic capillary column. Col-
`umn monolithic PS/DVB (50 ” 0.2 mm i.d.); mobile phase: (A) 100 mM
`TEAA, 0.1 mM Na4EDTA, pH 7.0, (B) 100 mM TEAA, 0.1 mM Na4EDTA,
`pH 7.0, 25 % acetonitrile; linear gradient: 0±5 % B in 1 min, 5±15 % B in
`2 min, 15±25 % B in 8 min, 25±30 % B in 4 min; flow rate: 3.0 ll/min; temper-
`ature, 60 C; detection: UV, 254 nm;
`injection volume 150 nL; sample
`8±32 mer oligonucleotides, 0.5±1.7 ng/lL each [50].
`
`Eng. Life Sci. 2005, 5, No. 5
`
`http://www.els-journal.de
`
` 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`433
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`Sci. Full Paper
`
`lower detection limits ranging from 0.5 to 3 fmol depending
`on the attached fluorophore. This means that a significant
`saving in PCR reagent consumption is achieved as amplifica-
`tions can be carried out in volumes as small as 0.5±1 lL.
`The chromatographic separation system can be readily on-
`line hyphenated to ESI-MS. Therefore, e.g., acetonitrile
`must be added post-column as sheath liquid through the
`electrospray ion source [51]. Furthermore, the concentration
`of the ion-pair reagent has to be reduced from 100 to 25 mM,
`which results in an additional improvement of analyte de-
`tectability.
`
`4 Analysis of Double-stranded DNA Fragments
`
`The permanent progresses in DNA technology, including
`DNA-cloning techniques, DNA restriction analysis, DNA
`sequencing, in-situ hybridization, polymerase chain reaction
`(PCR), and mutation-screening methods demand fast and
`sensitive analytical methods for the characterization of dou-
`ble-stranded DNA molecules.
`Fig. 4 shows the separation of 30 double-stranded DNA
`restriction fragments using a 50 ” 0.2 mm i.d. monolithic cap-
`illary column coming from pBr322 DNA-Hae III and a
`pBR322 DNA-Msp digest. Using a 200 lm i.d. capillary col-
`umn allows the reduction of sample amount by a factor of
`about 120 compared to a conventional column, which means
`that only about 2 fmol of the individual DNA fragments
`have to be injected using UV-absorbance detection at
`254 nm. The peak widths at half height were between 2.2
`and 4.5 s for the fragments up to a length of 217 bp, and be-
`tween 5.4 and 8.9 s for the longer fragments with a higher re-
`solving power compared to conventional HPLC [51].
`Coupling to ESI-MS provides information about the size
`of the DNA-fragment and allows a confident identification
`and characterization.
`
`5 Denaturing HPLC for Mutation Detection
`
`Partially denaturing HPLC exploits the reduced retention of
`heteroduplex molecules containing one or more mismatches
`as short deletions or insertions at elevated column tempera-
`tures compared to corresponding correctly matched homo-
`duplex over a temperature range of several degrees [16].
`DNA fragments shorter than approximately 150 bp are too
`unstable to allow the detection of mutations by partially de-
`naturing HPLC. This limitation can be overcome by the
`complete on-line denaturation of short amplicons into their
`single-stranded components, which are then resolved on the
`basis of their sequence composition rather than their size.
`As shown in Fig. 5, at a temperature of 56 C a successful
`detection of an A to G transition in a 209 bp amplicon can
`be carried out using both a monolithic and an encapsulated
`capillary column. The presence of a mismatch is recognized
`by the appearance of one or more additional peaks. The
`number of peaks detected is a function of the nature and the
`number of mismatches. The elution order is determined by
`the degree of destabilization of the DNA helix, which is in-
`fluenced by neighbouring stacking interactions [52] and the
`ability to form atypical Watson-Crick base pairs [53]. Using
`monolithic or encapsulated capillary columns lower concen-
`
`2
`
`1
`
`0
`
`2
`
`1
`
`0
`
`UV, 254 nm
`mV
`
`a
`
`b
`
`Figure 4. High-resolution IP-RP-HPLC of double-stranded DNA fragments
`obtained from HaeIII and MspI digests of pUC18 and pBR322 using a 0.2 mm
`i.d. monolithic capillary column. Column monolithic PS/DVB (50 ” 0.2 mm
`i.d.); mobile phase: (A) 100 mM TEAA, 0.1 mM Na4EDTA, pH 7.0,
`(B) 100 mM TEAA, 0.1 mM Na4EDTA, pH 7.0, 25 % acetonitrile; linear gra-
`dient: 30±50 % B in 4.0 min, 50±65 % B in 13 min; flow rate: 3.0 lL/min; tem-
`perature 49.7 C; detection: UV, 254 nm; injection volume 150 nL [50].
`
`5
`
`10
`
`time [min]
`
`15
`
`Figure 5. Mutation detection by DHPLC using (a) monolithic and (b) encap-
`sulated capillary. Column: (a) monolithic PS/DVB (50 ” 0.2 mm i.d.);
`(b) PS/DVB encapsulated Nucleosil (8 lm, 1000 Š); mobile phase: (A) 100 mM
`TEAA, 0.1 mM Na4EDTA, pH 7.0, (B) 100 mM TEAA, 0.1 mM Na4EDTA,
`pH 7.0, 25 % acetonitrile; linear gradient: 30±50 % B in 1.0 min, 50±70 % B in
`10 min; 70 % B for 5 min; flow rate: (a) 3.0 lL/min, (b) 2.0 mL/min; tempera-
`ture: (a) 57.8 C, (b) 55 C; detection: UV, 254 nm;
`injection volume:
`(a) 300 nL, (b) 500 nL.
`
`434
`
` 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
`
`http://www.els-journal.de
`
`Eng. Life Sci. 2005, 5, No. 5
`
`4
`
`

`

`trations of acetonitrile are required for the elution of the
`amplicons, which in turn results in higher stability of the
`double helical structure at a given temperature. A further
`advantage using capillary technology instead of conventional
`HPLC is the reduction of required sample size by a factor of
`approximately ten.
`An important feasibility of capillary IP-RP-HPLC is the
`possibility to identify heterozygous alleles eluting as one sin-
`gle chromatographic peak as ESI-MS can directly analyze
`and deconvolute mixtures of nucleic acids.
`
`6 Conclusions
`
`Monolithic and encapsulated separation media provide a
`great potential for the characterization of nucleic acids. The
`major advantage is based on the chromatographic separation
`efficiency caused by the optimized mass transfer properties
`due to the increased number of pore connectivity. The lack
`of retaining frits leads to a more rugged stationary phase ex-
`cluding the problems of bubble production and extra peak
`broadening. Due to the low inner diameter of 200 lm these
`phases show favourable sensitivity compared to convention-
`ally used HPLC columns. The hyphenation to ESI-MS al-
`lows to get important information about the identity and
`structure of nucleic acids for the identification of sequence
`variations in genomic DNA.
`
`Received: August 8, 2005 [ELS 63]
`Received in revised form: September 9, 2005
`Accepted: September 27, 2005
`
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